1. Field of the Invention
The present invention relates to peptides corresponding to fragments derived from the amino acid sequence of the 41 kD subunit protein constituting the fimbriae of Porphyomonas gingivalis, and their uses. More specifically, the invention relates to peptides acting as antigens which antigen-antibody react with antibodies against the 41 kD subunit protein, and uses of the peptides for detection of specific antibodies, etc. in the serum, saliva and gingival crevice fluid of patients with periodontal disease, and for prophylactic agents and treating agents of periodontal disease.
2. Description of Related Art
Periodontal diseases are classified into gingivitis and periodontitis, and further, periodontitis includes adult periodontitis, localized juvenile periodontitis, etc., but actually, 90% or more of periodontitis is occupied by adult periodontitis. These periodontal diseases are diseases including inflammations of gingiva, bleeding, drainage, formation of periodontal pockets, destruction of periodontal membranes, absorption of alveolar bones, and lability or loss of teeth. Various bacteria exist at the lesions of these periodontal diseases, and among them, Porphyomonas gingivalis is considered to be a main periodontopathic organism, and remarkable increase of the bacterium is observed at the lesions of periodontal diseases particularly adult periodontitis.
At present, methods for treating periodontal disease are not perfectly established, and it is considered to be the most important to find periodontal disease as early as possible, grasp its pathologic states accurately, and make appropriate treatments, and further, prophylaxis of periodontal disease by development of vaccines for periodontal disease is desired.
First, as to diagnosis of periodontal disease, a diagnostic method for finding periodontal disease as early as possible and grasping its pathologic state accurately has been expected, but actually, reliable diagnostic drugs for periodontal disease has not yet been developed. However, if ventured to be mentioned, as a means for diagnosing periodontal disease through paying attention to periodontopathic organisms, there are various methods for knowing the presence or number of these periodontopathic organisms. Further, there have been proposed methods to assay a specific antibody against a periodontopathic organism and utilize the result for diagnosis of periodontal disease. Still further, methods have been proposed comprising assaying an inflammatory product in the gingival crevice fluid of a patient with periodontal disease.
There are various methods for knowing the presence or number of these periodontopathic organisms. For example, it was conducted to culture periodontopathic organisms in the dental plaques, gingival crevice fluid, saliva, etc. of a patient with periodontal disease in a blood agar medium under an anaerobic condition, and investigate detailed biochemical properties of the resultant various colonies, and thereby detect the periodontopathic organisms (Loesche, W. J., Syed, S. A., Schmidt, E. and Morrison, E. C.: J. Periodont. 56, 447-456, 1985, etc.).
There has been conducted Gram staining, under microscopic observation, of periodontopathic organisms in dental plaques, gingival crevice fluid, saliva, etc. of a patient with periodontal diseases, or there has been made an examination under a dark-field microscope (Listgarten, M. A. and Levin, S.: J. Clin. Periodontol. 8, 122-138, 1981, etc.). There has also been carried out the detection of a periodonto-pathic organism by combining an antibody against it with a fluorescent dye (Zambon, J. J., Bochacki, V. and Genco, R. J.: Oral Microbiol. Immunol. 1, 39-44, 1986).
Further, it has been conducted to detect periodontopathic organisms in the dental plaques, gingival crevice fluid, saliva, etc. of a patient with a periodontal disease, according to an immunological method such as enzyme-linked immunosorbent assay (ELISA method), using antibodies against the respective periodontopathic organisms (see, Zambon, J. J., Bochacki, V. and Genco, R. J.: Oral Microbiol. Immunol. 1, 39-44, 1986; Japanese Laid-open Patent Publication No. 159762/1988; ibid. 107970/1990; ibid. 212061/1992; ibid. 355339/1992; ibid. 10954/1993; and EP 239,776 A).
Further, it has been conducted to pay attention to enzymes produced by periodontopathic organisms, and detect the periodontopathic organisms in the dental plaques, gingival crevice fluid, saliva, etc. of a patient with periodontal disease, using their enzymatic activities as an index (see Loesche, W. J., Oral Microbiol. Immunol. 1, 65-70, 1986; Japanese Laid-open Patent Publication No. 144997/1989; ibid. 144998/1989; ibid. 499/1990; and ibid. 229198/1992).
Recently, it has also been conducted to pay attention to genes of periodontopathic organisms, and detect the periodontopathic organisms in the dental plaques, gingival crevice fluid, saliva, etc. of a patient with periodontal disease, using a DNA probe method (DMDx (trademark), Biotechnica Diagnostics Co.; and see Japanese Laid-open Patent Publication No. 135096/1990; and ibid. 502640/1991). Further, it is becoming possible to detect an extremely slight amount of cells, according to the PCR (polymerase chain reaction) method.
However, diagnostic methods, as above-mentioned, for periodontal disease to know the presence and number of periodontopathic organisms are usable only for diagnosis of pathogenesis, and are insufficient for grasping the pathologic state of a patient. As a diagnostic method which makes a pair therewith, and is for knowing the response of a patient as a host to periodontopathic organisms and thereby grasping the pathologic state of the patient, there can be thought a diagnostic method for periodontal disease to know a specific antibody against a periodontopathic organism, and further its class and subclass. A diagnostic method for periodontal disease to know the presence and number of periodontopathic organisms is also, of course, important, and it is said that it is important to conduct appropriate diagnosis of periodontal disease by combining both methods.
As one of such diagnostic methods, a method is proposed to assay the specific antibody titer against a periodontopathic organism of a patient with adult periodontitis by the ELISA method, and diagnose the periodontal disease of the patient from both of the results and the results of dental checkups (see Mouton, C., et al.: Infection and Immunity 31, 182-192, 1981; Japanese Laid-open Patent Publication No. 162753/1986; and ibid. 107969/1990).
Further, recently, a possibility that a diagnostic method for periodontal disease is effective based on not only increase of a specific antibody against a periodontopathic organism of a patient with periodontal disease, but the fact that the subclass of the specific antibody changes from IgG.sub.1 to IgG.sub.4 together with change of the pathologic state of the periodontal disease is proved by the ELISPOT method to detect antibody-producing cells against periodontopathic organisms in the gingiva tissue of a patient of adult periodontitis (T. Ogawa et al.: Clin. exp. Immunol. 76, 103-110, 1989).
When increase of a specific antibody against a periodontopathic organism of a patient with periodontal disease, and further change of its class or subclass are checked, an antigen used for detection becomes necessary. In the above usual methods, entire cells of a periodontopathic organism, or cell surface components such as lipopolysaccharides and fimbriae as an antigen have been used as antigens. However, it is difficult to obtain a large amount of pure antigen by the procedure of separation and purification, and many non-relevant reactions take place thereon, and thus, they are not desirable for accurately checking increase of specific antibodies against the periodontopathic organisms of a patient with periodontal disease, and further their classes or subclasses.
BBRC. 180, No. 3, 1335-1341 (1991) reports the immunogenicity of a synthetic peptide composed of 20 amino acid residues in the 41 kD subunit protein molecule constituting the fimbriae of Porphyomonas gingivalis.
Further, it is disclosed in Japanese Laid-open Patent Publication No. 135096/1990 to use a component derived from a periodontopathic organism in a patient with periodontal disease for detecting a specific antibody against the periodontopathic organism, but the antigenic protein derived from Porphyomonas gingivalis disclosed therein utterly differs from the fimbria of Porphyomonas gingivalis as a substance, and further, is not desirable because it is troublesome to prepare an antigen as an entire protein. As one of other diagnostic methods for periodontal disease, there is a method to assay an inflammatory product. For example, a method is proposed to assay collagenase (Periocheck (trademark): Advanced Clinical Technologies, Westwood, Mass.), a cathepsin-like activity (Progno Stick: Dentsply Corp., York Pa.), or glucuronidase (Abbott Laboratories, North Chicago, Ill.). However, these methods have problems in specificity.
In Japanese Patent Publication No. 13205/1991, Japanese laid-open Patent Publication No. 140527/1986 and Japanese laid-open Patent Publication No. 132428/1993, it is proposed to use the fimbriae of Porphyomonas gingivalis as a prophylactic vaccine against periodontitis (or periodontal disease). However, there are many problems in using fimbriae separated and purified according to methods mentioned therein. Namely, when the fimbriae are used after separation and purification, it is thought that substances other than the fimbriae, for example, cellular components other than the fimbriae such as lipopolysaccharides which are pyrogens mingle therein, and the fimbriae are not desirable as vaccines. As understood from that it is shown in BBRC. 180, No. 3, 1335-1341 (1991) that these fimbriae themselves exhibit various biological activities, there is a possibility that use of whole of these fimbriae causes actions other than the action as a vaccine, for example, a prophlogistic action and a pyrogenic action, which is undesirable.
Incidentally, the antigenic protein derived from Porphyomonas gingivalis, which is exhibited to be used as a vaccine in Japanese Patent Publication No. 135096/1990, differs from the peptide of the fimbriae of Porphyomonas gingivalis in the invention in amino acid sequence, and it is not desirable to use the entire antigenic protein because preparation of the vaccine is troublesome and there is a possibility that it causes actions other than the action as a vaccine, for example, a prophlogistic action and a pyrogenic action. Further, the synthetic peptide of the fimbriae of Porphyomonas gingivalis shown in Adv. Exp. Med. Biol. 327, 255-262, 1992 differs from the peptide of the fimbriae of Porphyomonas gingivalis in the invention in region, and it is not desirable, as in the above case, to use the entire protein because preparation of the vaccine is troublesome and there is a possibility that it causes actions other than the action as a vaccine, for example, a prophlogistic action and a pyrogenic action.
An oral cavity composition containing an antibody obtained by immunization with the whole cell or fimbrial protein of Porphyomonas gingivalis is proposed in Japanese laid-open Patent Publication No. 142915/1985, ibid. 277632/1986, ibid. 289024/1986, ibid. 417/1987, ibid. 313438/1989, ibid. 53458/1990, ibid. 53716/1990, ibid. 218620/1990, ibid. 59736/1992, ibid. 59737/1992 and Japanese Patent Publication No. 63865/1992. However, antibodies against the whole cell or fimbrial protein prepared by these methods possibly cause cross reaction with substances other than the desired antigen, and are not desirable for the present objects.
Thus, the object of the invention lies in providing peptides capable of effective detection of increase of specific antibodies against periodontopathic organisms in the patient with periodontal disease, and further change of their classes or subclasses, and a composition such as a peptide for diagnosis of periodontal disease. Another object of the invention lies in providing a vaccine for prophylaxis of periodontal disease containing a peptide having substantially no side-effect other than the desired vaccine action, among these peptides, and an oral cavity composition for prophyl axis or treatment of periodontal disease containing an antibody obtained by immunizing an animal with a peptide thus selected.